NMR Dynamics Investigation of Ligand-Induced Changes of Main and Side-Chain Arginine N-H’s in Human Phosphomevalonate Kinase

Phosphomevalonate kinase (PMK) catalyzes phosphoryl transfer from adenosine triphosphate (ATP) to mevalonate 5-phosphate (M5P) on the pathway for synthesizing cholesterol and other isoprenoids. To permit this reaction, its substrates must be brought proximal, which would result in a significant and repulsive buildup of negative charge. To facilitate this difficult task, PMK contains 17 arginines and eight lysines. However, the way in which this charge neutralization and binding is achieved, from a structural and dynamics perspective, is not known. More broadly, the role of arginine side-chain dynamics in binding of charged substrates has not been experimentally defined for any protein to date. Herein we report a characterization of changes to the dynamical state of the arginine side chains in PMK due to binding of its highly charged substrates, ATP and M5P. These studies were facilitated by the use of arginine-selective labeling to eliminate spectral overlap. Model-free analysis indicated that while substrate binding has little effect on the arginine backbone dynamics, binding of either substrate leads to significant rigidification of the arginine side chains throughout the protein, even those that are \u3e8 Å from the binding site. Such a global rigidification of arginine side chains is unprecedented and suggests that there are long-range electrostatic interactions of sufficient strength to restrict the motion of arginine side chains on the picosecond-to-nanosecond time scale. It will be interesting to see whether such effects are general for arginine residues in proteins that bind highly charged substrates, once additional studies of arginine side-chain dynamics are reported

Substrate Induced Structural and Dynamics Changes in Human Phosphomevalonate Iinase and Implications for Mechanism

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the γ-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg-ADP, M5P, and the ternary complex) in PMK using NMR-based dynamics and chemical shift perturbation measurements. Mg-ADP and M5P Kd\u27s were 6–60 μM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (τc = 13.5 nsec), whereas subsequent binding of Mg-ADP opens the structure up (τc = 15.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S2 ∼0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the “Walker-A” catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for γ-phosphate binding interactions. Indeed, the role of γ-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the β-γ bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N-terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence. Proteins 2009. © 2008 Wiley-Liss, Inc

Molecular Docking and NMR Binding Studies to Identify Novel Inhibitors of Human Phosphomevalonate Kinase

Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based 1H–15N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (Kd). Tight binding compounds with Kd’s ranging from 6–60 μM were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development

Local structural plasticity of the Staphylococcus aureus evasion protein EapH1 enables engagement with multiple neutrophil serine proteases

Members of the EAP family of Staphylococcus aureus immune evasion proteins potently inhibit the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin-G, and proteinase-3. Previously, we determined a 1.8 A resolution crystal structure of the EAP family member EapH1 bound to neutrophil elastase. This structure revealed that EapH1 blocks access to the enzyme’s active site by forming a noncovalent complex with this host protease. To determine how EapH1 inhibits other NSPs, we studied here the effects of EapH1 on cathepsin-G. We found that EapH1 inhibits cathepsin-G with a Ki of 9.8 ± 4.7 nM. Although this Ki value is ~466-fold weaker than the Ki for EapH1 inhibition of neutrophil elastase, the time dependence of inhibition was maintained. To define the physical basis for EapH1’s inhibition of cathepsin-G, we crystallized EapH1 bound to this protease, solved the structure at 1.6 A resolution, and refined the model to Rwork and Rfree values of 17.4% and 20.9%, respectively. This structure revealed a protease-binding mode for EapH1 with cathepsin-G that was globally similar to that seen in the previously determined EapH1–neutrophil elastase structure. The nature of the intermolecular interactions formed by EapH1 with cathepsin-G differed considerably from that with neutrophil elastase, however, with far greater contributions from the inhibitor backbone in the cathepsin-G– bound form. Together, these results reveal that EapH1’s ability to form high-affinity interactions with multiple NSP targets is due to its remarkable level of local structural plasticity

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH–rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery